Peroxiredoxin-2 recycling is slower in denser and pediatric sickle cell red cells.

Peroxiredoxin-2 (Prx-2) is a critical antioxidant protein in red blood cells (RBC). Prx-2 is oxidized to a disulfide covalently-bound dimer by H2 O2 , and then reduced back by the NADPH-dependent thioredoxin-thioredoxin reductase system. The reduction of oxidized Prx-2 is relatively slow in RBCs. Since Prx-2 is highly abundant, Prx-2s' peroxidase catalytic cycle is not considered to be limiting under normal conditions. However, whether Prx-2 recycling becomes limiting when RBCs are exposed to stress is not known. Using three different model systems characterized by increased oxidative damage to RBCs spanning the physiologic (endogenous RBCs of different ages), therapeutic (cold-stored RBCs in blood banks) and pathologic (RBCs from sickle cell disease (SCD) patients and humanized SCD mice) spectrum, basal levels of Prx-2 oxidation and Prx-2 recycling kinetics after addition of H2 O2 were determined. The reduction of oxidized Prx-2 was significantly slower in older versuin older versus younger RBCs, in RBCs stored for 4-5 weeks compared to 1 week, and in RBC from pediatric SCD patients compared to RBCs from control non-SCD patients. Similarly, the rate of Prx-2 recycling was slower in humanized SCD mice compared to WT mice. Treatment of RBC with carbon monoxide (CO) to limit heme-peroxidase activity had no effect on Prx-2 recycling kinetics. Treatment with glucose attenuated slowed Prx-2 recycling in older RBCs and SCD RBCs, but not stored RBCs. In conclusion, the reduction of oxidized Prx-2 can be further slowed in RBCs, which may limit the protection afforded by this antioxidant protein in settings associated with erythrocyte stress.

A large multi-focus dataset for white blood cell classification.

The White Blood Cell (WBC) differential test ranks as the second most frequently performed diagnostic assay. It requires manual confirmation of the peripheral blood smear by experts to identify signs of abnormalities. Automated digital microscopy has emerged as a solution to reduce this labor-intensive process and improve efficiency. Several publicly available datasets provide various WBC subtypes of differing quality and resolution. These datasets have contributed to advancing WBC classification using machine learning techniques. However, digital microscopy of blood cells with high magnification often requires a wider depth of field, posing challenges for automatic digital microscopy that necessitates capturing multiple stacks of focal planes to obtain complete images of specific blood cells. Our dataset provides 25,773 image stacks from 72 patients. The image labels consist of 18 classes encompassing normal and abnormal cells, with two experts reviewing each label. Each image includes 10 z-stacks of cropped 200 by 200 pixel images, captured using a 50X microscope with 400 nm intervals. This study presents a comprehensive multi-focus dataset for WBC classification.

Embedded-deep-learning-based sample-to-answer device for on-site malaria diagnosis.

Improvements in digital microscopy are critical for the development of a malaria diagnosis method that is accurate at the cellular level and exhibits satisfactory clinical performance. Digital microscopy can be enhanced by improving deep learning algorithms and achieving consistent staining results. In this study, a novel miLab™ device incorporating the solid hydrogel staining method was proposed for consistent blood film preparation, eliminating the use of complex equipment and liquid reagent maintenance. The miLab™ ensures consistent, high-quality, and reproducible blood films across various hematocrits by leveraging deformable staining patches. Embedded-deep-learning-enabled miLab™ was utilized to detect and classify malarial parasites from autofocused images of stained blood cells using an internal optical system. The results of this method were consistent with manual microscopy images. This method not only minimizes human error but also facilitates remote assistance and review by experts through digital image transmission. This method can set a new paradigm for on-site malaria diagnosis. The miLab™ algorithm for malaria detection achieved a total accuracy of 98.86% for infected red blood cell (RBC) classification. Clinical validation performed in Malawi demonstrated an overall percent agreement of 92.21%. Based on these results, miLab™ can become a reliable and efficient tool for decentralized malaria diagnosis.

In situ analysis of heterogeneity in the lipid content of single green microalgae in alginate hydrogel microcapsules.

Microalgae, a group of microorganisms that grow using sunlight as the sole energy source and carbon dioxide as an only carbon source, have been considered as a feedstock of choice for the production of biofuels such as biodiesel. To explore the economic feasibility of such application, however, many technical hurdles must first be overcome; the selection and/or screening of competent species are some of the most important and yet challenging tasks. To greatly accelerate this rather slow and laborious step, we developed a droplet-based microfluidic system that uses alginate hydrogel microcapsules with a mean diameter of 26 µm, each of which is able to encapsulate a single microalgal cell. This novel device was successfully demonstrated using three microalgae species, namely, Chlorella vulgaris , Chlamydomonas sp., and Botryococcus braunii . In situ analysis of the lipid content of individual microalgal cells by nondestructive fluorescence staining using BODIPY (4,4-difluoro-1,3,5,7,-tetramethyl-4-bora-3a,4a-diaza-s-indacene) was possible. In all cases, we confirmed that the lipid content of microalgal species in alginate hydrogel microcapsules was comparable to that of free-living cells. Stochastic heterogeneity in the lipid content was verified under a highly viable physiological condition, implying that other analyses were possible after the determination of lipid content. Furthermore, the designed microwell arrays enabled us to distinguish the BODIPY fluorescence response of a single live alga within the microcapsules.

Label-free cancer cell separation from human whole blood using inertial microfluidics at low shear stress.

We report a contraction-expansion array (CEA) microchannel device that performs label-free high-throughput separation of cancer cells from whole blood at low Reynolds number (Re). The CEA microfluidic device utilizes hydrodynamic field effect for cancer cell separation, two kinds of inertial effects: (1) inertial lift force and (2) Dean flow, which results in label-free size-based separation with high throughput. To avoid cell damages potentially caused by high shear stress in conventional inertial separation techniques, the CEA microfluidic device isolates the cells with low operational Re, maintaining high-throughput separation, using nondiluted whole blood samples (hematocrit ~45%). We characterized inertial particle migration and investigated the migration of blood cells and various cancer cells (MCF-7, SK-BR-3, and HCC70) in the CEA microchannel. The separation of cancer cells from whole blood was demonstrated with a cancer cell recovery rate of 99.1%, a blood cell rejection ratio of 88.9%, and a throughput of 1.1 × 10(8) cells/min. In addition, the blood cell rejection ratio was further improved to 97.3% by a two-step filtration process with two devices connected in series.

Quantification of solution-free red blood cell staining by sorption kinetics of Romanowsky stains to agarose gels.

The imaging and quantification of stained red blood cells (RBCs) are important for identifying RBCs in hematology and for diagnosing diseased RBCs or parasites in cytopathology. Romanowsky staining has been used traditionally to produce hues in blood cells using a mixture of anionic eosin Y and cationic methylene blue and azure B. While Romanowsky stains have been widely used in cytopathology, end-users have experienced problems with varying results in staining due to the premature precipitation or evaporation of methanol, leading to the inherent inconsistency of solution-based Romanowsky staining. Herein, we demonstrate that the staining and destaining of blood smears are controllable by the contact time of agarose gel stamps. While the extent of staining and destaining is discernable by the hue values of stamped red blood cells in micrographs, the quantification of adsorbed and desorbed Romanowsky dye molecules (in particular, eosin Y, methylene blue and azure B) from and to the agarose gel stamps needs a model that can explain the sorption process. We found predictable sorption of the Romanowsky dye molecules from the pseudo-second-order kinetic model for adsorption and the one phase decay model for desorption. Thus, the method of agarose gel stamping demonstrated here could be an alternative to solution-based Romanowsky staining with the predictable quantity of sorption and timing of contact.

Self-reference quantitative phase microscopy for microfluidic devices.

This Letter describes a quantitative phase microscopy for microfluidic devices using a simple self-referencing interferometry. Compared with the gross dimensions of the microfluidic device, the microchannel occupies only a small area of the device. Hence, the reference field can be generated by inverting the relative position of the specimen and background. Our system is realized using an extended depth-of-field optics in the form of Michelson interferometry, which allows quantitative phase measurement for an increased depth-of-field without moving objective lens or specimen. Furthermore, the system can be readily converted to a higher signal-to-noise ratio Hilbert phase microscopy thanks to the simultaneous acquisition of double interferograms. The performance of our system is verified using polymer beads, micropatterning poly(dimethylsiloxane) (PDMS), and embryo cells in the microchannels.

A microfluidic in vitro cultivation system for mechanical stimulation of bovine embryos.

This work demonstrates a novel microfluidic in vitro cultivation system for embryos that improves their development using a partially constricted channel that mimics peristaltic muscle contraction. Conventional photolithography and a PDMS replica molding process were used to make straight or constricted microchannels. To investigate the effects of constriction geometry on embryonic development, different constriction widths of the channel were designed. Bovine embryos were loaded and incubated by simply placing them on a tilting machine to provide embryo movement via gravity. The fertilized embryos were cultivated on the microfluidic in vitro cultivation system until the blastocyst, hatching, or hatched blastocyst stages. To confirm the quality of blastocysts in the microfluidic channel, double staining was performed and compared with bovine embryos cultivated by the conventional droplet method. The proportion of eight-cell development among total embryos in the constricted channel (56.7+/-13.7%; mean+/-SD) was superior to that in the straight channel (23.9+/-11.0%). This suggests that the effect of constriction is vital for the early development of bovine embryos in assisted-reproduction research.

Demonstration of Interposed Modular Hydrogel Sheet for Multicellular Analysis in a Microfluidic Assembly Platform.

Hydrogel sheets have emerged as a promising biomaterial scaffold for the encapsulation and transfer of multicellular structures. Although the improvement of the chemical interactions and the design of micro-scaled geometry have contributed to the development of multipurpose hydrogel scaffolds, the application of hydrogel sheets to assess multicellular structures is still challenging. To expand the technical applicability of hydrogel sheets, we here demonstrate that a single layer of the hydrogel sheet can be integrated as an interposed module in a microfluidic device for multicellular analysis. As a cell culture unit, encapsulated pancreatic insulinoma (MIN6) cells in the hydrogel sheet were labeled and examined via multiple microchannels. After obtaining simultaneously multi-labeled cells in the hydrogel sheet that had been incorporated into the microfluidic device, each modular hydrogel sheet was also recoverable and re-cultured without any distortion. The modular hydrogel sheet can be simply manipulated and conserved as a multicellular module in a three-dimensional (3D) in vitro culture platform. Using the modular concept of hydrogel sheets capable of cell culture and/or assay, an integrated multicellular analysis in the microfluidic device is expected to improve accessibility, scalability, and practicality for end users.

Tolerance and pharmacokinetics of a ciprofloxacin-coated sinus stent in a preclinical model.

BACKGROUND: Chronic rhinosinusitis (CRS) is often associated with persistent bacterial infection despite the use of systemic antibiotics. Topically administered antibiotics are an alternative strategy, but require effective local concentrations, prolonged mucosal contact time, minor systemic absorption, and minimal depletion. The objectives of the current study were to analyze the in vitro release rate and in vivo drug delivery tolerance and pharmacokinetics of a ciprofloxacin-coated sinus stent (CSS). METHODS: The CSS (2 mg) was created from biodegradable poly-D/L-lactic acid. After analyzing in vitro release profile, CSSs were placed unilaterally in maxillary sinuses of 16 rabbits via dorsal sinusotomy. Animals were euthanized between 1 and 3 weeks postoperatively. Ciprofloxacin concentrations in the sinus tissue and plasmas were assessed using high-performance liquid chromatography. Radiological and histological evaluations were performed. RESULTS: In the in vitro release profile, an initial burst release was observed over the first 24 hours, followed by sustained release through the 14-day time point. In the rabbit model, ciprofloxacin was continuously released from the stent up to 3 weeks at doses >50 ng/mL. Histologic examination found no evidence of inflammation, epithelial ulceration, or bony reaction upon euthanization of the animals at 21 days. Computed tomography also demonstrated no signs of mucosal edema or opacification in the sinus. CONCLUSION: The CSS was safe in this preclinical model and sustained release was observed in both the in vitro and in vivo analyses. The innovative stent design coated with ciprofloxacin may provide a unique therapeutic strategy for chronic rhinosinusitis (CRS).

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture.

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 µm, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

Freestanding stacked mesh-like hydrogel sheets enable the creation of complex macroscale cellular scaffolds.

Hydrogel-based bottom-up tissue engineering depends on assembly of cell-laden modules for complex three-dimensional tissue reconstruction. Though sheet-like hydrogel modules enable rapid and controllable assembly, they have limitations in generating spatial microenvironments and mass transport. Here, we describe a simple method for forming large-scale cell-hydrogel assemblies via stacking cell-embedded mesh-like hydrogel sheets to create complex macroscale cellular scaffolds. Freestanding stacked hydrogel sheets were fabricated for long-term cell culturing applications using a facile stacking process where the micropatterned hydrogel sheets (8.0 mm × 8.7 mm) were aligned using a polydimethylsiloxane drainage well. The stacked hydrogel sheets were precisely aligned so that the openings could facilitate mass transport through the stacked sheets. Despite the relatively large height of the stacked structure (400-700 µm), which is larger than the diffusion limit thickness of 150-200 µm, the freestanding cell-ydrogel assemblies maintained cell viability and exhibited enhanced cellular function compared with single hydrogel sheets. Furthermore, a three-dimensional co-culture system was constructed simply by stacking different cell-containing hydrogel sheets. These results show that stacked hydrogel sheets have significant potential as a macroscale cell-culture and assay platform with complex microenvironments for biologically relevant in vitro tissue-level drug assays and physiological studies.

Progress and challenges of the bioartificial pancreas.

Pancreatic islet transplantation has been validated as a treatment for type 1 diabetes since it maintains consistent and sustained type 1 diabetes reversal. However, one of the major challenges in pancreatic islet transplantation is the body's natural immune response to the implanted islets. Immunosuppressive drug treatment is the most popular immunomodulatory approach for islet graft survival. However, administration of immunosuppressive drugs gives rise to negative side effects, and long-term effects are not clearly understood. A bioartificial pancreas is a therapeutic approach to enable pancreatic islet transplantation without or with minimal immune suppression. The bioartificial pancreas encapsulates the pancreatic islets in a semi-permeable environment which protects islets from the body's immune responses, while allowing the permeation of insulin, oxygen, nutrients, and waste. Many groups have developed various types of the bioartificial pancreas and tested their efficacy in animal models. However, the clinical application of the bioartificial pancreas still requires further investigation. In this review, we discuss several types of bioartificial pancreases and address their advantages and limitations. We also discuss recent advances in bioartificial pancreas applications with microfluidic or micropatterning technology.

User-friendly 3D bioassays with cell-containing hydrogel modules: narrowing the gap between microfluidic bioassays and clinical end-users' needs.

Cell-containing hydrogel modules as cell-hydrogel microunits for creating a physiologically relevant 3D in vivo-like microenvironment with multiple cell types and unique extracellular matrix (ECM) compositions facilitate long-term cell maintenance and bioassays. To date, there have been many important advances in microfluidic bioassays, which incorporate hydrogel scaffolds into surface-accessible microchambers, driven by the strong demand for the application of spatiotemporally defined biochemical stimuli to construct in vivo-like conditions and perform real-time imaging of cell-matrix interactions. In keeping with the trend of fostering collaborations among biologists, clinicians, and microfluidic engineers, it is essential to create a simpler approach for coupling cell-containing hydrogel modules and an automated bioassay platform in a user-friendly format. In this article, we review recent progress in hydrogel-incorporated microfluidics for long-term cell maintenance and discuss some of the simpler and user-friendly 3D bioassay techniques combined with cell-containing hydrogel modules that can be applied to mutually beneficial collaborations with non-engineers. We anticipate that this modular and user-friendly format interfaced with existing laboratory infrastructure will help address several clinical questions in ways that extend well beyond the current 2D cell-culture systems.

Geometric effect of the hydrogel grid structure on in vitro formation of homogeneous MIN6 cell clusters.

A microstructure-based hydrogel was employed to study the relationship between spatial specificity and cellular behavior, including cell fate, proliferation, morphology, and insulin secretion in pancreatic ß-cells. To effectively form homogeneous cell clusters in vitro, we made cell-containing hydrogel membrane constructs with an adapted grid structure based on a hexagonal micropattern. Homogeneous cell clusters (average diameter: 83.6 ± 14.2 µm) of pancreatic insulinoma (MIN6) cells were spontaneously generated in the floating hydrogel membrane constructs, including a hexagonal grid structure (size of cavity: 100 µm, interval between cavities: 30 µm). Interestingly, 3D clustering of MIN6 cells mimicking the structure of pancreatic islets was coalesced into a merged aggregate attaching to each hexagonal cavity of the hydrogel grid structure. The fate and insulin secretion of homogeneous cell clusters in the hydrogel grid structure were also assessed. The results of these designable hydrogel-cell membrane constructs suggest that facultative in vitro ß-cell proliferation and maintenance can be applied to biofunctional assessments.

Cellular hydrogel biopaper for patterned 3D cell culture and modular tissue reconstruction.

Microarchitectured freestanding cellular hydrogel biopaper as a novel 3D cell culture or tissue reconstruction module is reported. New harvesting, transfer, and assembly techniques are used to construct laminated tissue composites of the biopaper, such as hepatic hydrogel sheet modules with augmented liver function for stratified 3D hepatic tissue reconstruction.

Breast cancer diagnosis using a microfluidic multiplexed immunohistochemistry platform.

BACKGROUND: Biomarkers play a key role in risk assessment, assessing treatment response, and detecting recurrence and the investigation of multiple biomarkers may also prove useful in accurate prediction and prognosis of cancers. Immunohistochemistry (IHC) has been a major diagnostic tool to identify therapeutic biomarkers and to subclassify breast cancer patients. However, there is no suitable IHC platform for multiplex assay toward personalized cancer therapy. Here, we report a microfluidics-based multiplexed IHC (MMIHC) platform that significantly improves IHC performance in reduction of time and tissue consumption, quantification, consistency, sensitivity, specificity and cost-effectiveness. METHODOLOGY/PRINCIPAL FINDINGS: By creating a simple and robust interface between the device and human breast tissue samples, we not only applied conventional thin-section tissues into on-chip without any additional modification process, but also attained perfect fluid control for various solutions, without any leakage, bubble formation, or cross-contamination. Four biomarkers, estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR) and Ki-67, were examined simultaneously on breast cancer cells and human breast cancer tissues. The MMIHC method improved immunoreaction, reducing time and reagent consumption. Moreover, it showed the availability of semi-quantitative analysis by comparing Western blot. Concordance study proved strong consensus between conventional whole-section analysis and MMIHC (n = 105, lowest Kendall's coefficient of concordance, 0.90). To demonstrate the suitability of MMIHC for scarce samples, it was also applied successfully to tissues from needle biopsies. CONCLUSIONS/SIGNIFICANCE: The microfluidic system, for the first time, was successfully applied to human clinical tissue samples and histopathological diagnosis was realized for breast cancers. Our results showing substantial agreement indicate that several cancer-related proteins can be simultaneously investigated on a single tumor section, giving clear advantages and technical advances over standard immunohistochemical method. This novel concept will enable histopathological diagnosis using numerous specific biomarkers at a time even for small-sized specimens, thus facilitating the individualization of cancer therapy.